The present invention is directed to a methodology for highly efficient, stable integration of DNA into a eukaryotic genome. More specifically, the present invention is directed to the use of a synthetic polypeptide, containing a nuclear localization signal to complex with a DNA molecule and to facilitate its transportation and integration into the nuclear genome of a mammalian or other eukaryotic cell, for example, in the context of producing cell lines with an extended life.
DNA-CaPO4 co-precipitation was the first method developed to introduce genes into mammalian cells. (“Gene” in this regard denotes a structural DNA segment, i.e., a DNA that codes for a polypeptide, and comprehends oncogenes as well as DNAs coding for a known expression product.) The co-precipitation method was applicable only to certain cell types, however, and could not be used to introduce genes into a wide variety of cell lines, especially those of hematopoietic origin. Moreover, the stable gene transfer efficiency was rather low, on the order of 10−4 to 10−6. McNally, M. A., et. al., BioTechniques 6: 8826 (1988); Yen, T. S. B., et. al., loc. cit. 6: 413 (1988).
Limits on introducing and expressing genes in cultured mammalian cells motivated a search for other, more efficient approaches to gene transfer. Methods were developed, for example, that utilized chemical agents which were positively charged and, hence, able to complex with negatively charged DNA molecules. Examples of such agents include DEAE dextran and various cationic lipid molecules. Cells treated with DNA complexes comprised of such an agent can lead to the introduction of the DNA into different mammalian cell lines. Mannino, R. J. et al., BioTechniques 6: 682 (1988); Felgner, P. et al., Proc. Nat'l Acad. Sci. USA 84: 7413 (1987); Fraley, R. et. al., Trend Biochem. Sci. 6: 77 (1981); Holter, W. et. al., Exp. Cell Res. 184: 546 (1989); McCutchan, J. H. et al., J. Nat'l Cancer Inst. 41: 351 (1986); Chaney, W. C. et al., Somatic Cell & Mol. Genet. 12: 237 (1986).
The production of a gene product for only a short time period after transfection, usually from 48 to 72 hours, is called “transient expression.” Many of the DNA-complexing agents reported heretofore, while useful in transferring a gene into mammalian cells, resulted in only transient expression of the introduced gene in a small fraction of the transfected cells. See, for example, Miller et. al., Proc. Nat'l Acad. Sci., USA, 76: 949 (1979); Oi et al., loc. cit. 80: 825 (1983).
In addition to giving poor results with respect to stable gene expression, transfer methods based on such DNA-complexing agents often were effective only with established cell lines, and did not work very well with primary cells isolated from various mammalian species. Other techniques therefore were needed to enhance gene transfer efficiency, to increase the variety of cell types capable of being transfected, and to effect stable gene transfer. Stable gene transfer is the ability of cells to maintain and express transfected DNAs in a stable manner, through integration of the transfected DNA into cell chromosomes.
Retrovital vectors, which were under development at about the same time seemed to be quite effective in transferring genes into different cell types. The use of such vectors was prompted by the elucidation of gene regulation in various murine and avian retroviruses. Two other developments led to the development of retrovirus-based gene transfer vehicles. The first development was the identification of minimal sequences required for efficient packaging of viral particles in a cell line which produced the coat proteins and other structural components of the viral particle in trans. The cell lines that provided the structural components for virus development are called “packaging” cell lines. The second significant step in the establishment of retroviral vectors was the development of both ecotropic and amphotropic packaging cell lines, which aided the design of recombinant retroviral particles which could infect both murine and human cell lines.
Additional modifications of retroviruses were deemed necessary to address concerns that retroviral vectors could recombine in vivo to generate wild-type virus. Developments in this regard yielded a number of safe retroviral vectors which have been used to transfer genes into a variety of established mammalian cell lines, as well as into certain primary cells in a few instances. E. Gilboa et al., BioTechniques 4: 504 (1986); A. D. Miller et al., Mol. Cell. Biol. 6: 2895 (1986); H. Stuhlmann et al., loc. cit. 9: 100 (1989); A. D. Miller et al., BioTechniques 7: 980 (1989); J. A. Zwiebel et al., Science 243: 220 (1989).
Even though these vectors were effective with respect to various mammalian cells, there were many restrictions on a wider application of the retroviral gene-transfer technique. These limitations included (1) the size of exogenous DNA that can be inserted into a retroviral vector and (2) the use of only dividing cells for retroviral gene transfer. E. Gilboa, BioTechniques, supra (1986); A. D. Miller, supra (1986); H. Stuhlmann, et al., Mol. Cell. Biol. supra, (1986); A. D. Miller et al., BioTechniques, supra (1986); J. A. Zwiebel et al., supra (1989).
Other viruses have been used to generate recombinant viral vectors for gene transfer studies. Adenovirus, adeno-associated virus, herpes simplex virus, and even HIV have been employed as vectors to introduce genes into both established cell lines and primary cells. Some of these viral vectors are capable of transferring genes into non-dividing cells. R. J. Samulski, et al., EMBO J. 10: 3941 (1981); J. D. Tratschin, et al., Mol. Cell. Biol. 5: 3251 (1985); P. L. Hermonat, et al., Proc. Nat'l Acad. Sci. (USA) 81: 6466 (1984); D. J. Fink, et al., Human Gene Therapy 3: 11 (1992).
Viral vectors capable of transferring genes into non-dividing cells usually require the generation of high-titer viral stock in order to achieve high efficiency gene transfer into different cell types. In addition, whenever a different regulatory sequence is to be tested for optimal level of gene expression into primary cells, a new viral stock must to be made and tittered for every modification. All these involve very time-consuming experimental manipulations.
Still another concern relates to the application of viral vectors in human gene therapy. A number of studies have been carried out in primates to test the safety of retroviral vectors for introducing cells transduced with retroviral vectors into animals. Some of these animals have developed various forms of lymphoma. R. E. Donahue, et al., J. Exp. Med. 176: 1125 (1992). Additional safety features have been introduced into some of the newer versions of retroviral vectors, yet are not available for all types of viral vectors.